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9/16/16 Enzymesareusedalloveryourbody! Biochemistry Whatisanenzyme? • Almostallenzymesareproteinsthatactas biologicalcatalysts. 3.1)Enzymes-I • Introductiontoenzymestructureand function,andfactorsinvolvingtheir actionsandpathways Acatalystspeedsupchemicalreactions.Enzymes speedupbiologicalchemicalreactions. • Enzymesarehighlyspecifictoatypeofreaction. • Enzymesmustmaintaintheirspecificshapeinorder tofunction.Anyalterationintheprimary, secondary,tertiary,orquaternaryformsofthe enzymearedetrimental. Prof. Dr. Klaus Heese EnzymesasBiologicalCatalysts CO2 +H2O--->H2CO3 • Enzymes areproteins thatincreasetherate ofreactionby loweringtheenergy ofactivation • Theycatalyzenearly allthechemical DG reactionstaking placeinthecellsof thebody • Enzymeshaveunique three-dimensional •Increasedreactionratessometimes106 to1012 increase shapesthatfitthe shapesofreactants EnzymesdonotchangeDG,justthereactionrates. (substrates) Enzymescatalyzebystabilizingtransitionstates • FreeenergyGofachemical reactioncanbeplottedovertime • Favorablereactionshavea positivedifference(DG)infree energybetweenthesubstrate andproduct • Thefreeenergyofactivationfor thetransitionstatelimitsthe progressofthereaction • Enzymesactbyreducingthefree energyofthetransitionstate (Enzymesalterrates,notequilibria) Functionofenzymes Enzymeshavemanyjobs.They: • Breakdownnutrientsintouseablemolecules. • Storeandreleaseenergy(ATP). • Createlargermoleculesfromsmallerones. • Coordinatebiologicalreactionsbetweendifferent systemsinanorganism. •Milderreactionconditions •Greatreactionspecificity Naming Enzymes • Thenameofanenzymeidentifiesthereactingsubstance - usuallyendsin–ase • Forexample,sucrase catalyzesthehydrolysisofsucrose • Thenamealsodescribesthefunctionoftheenzyme • Forexample,oxidases catalyzeoxidationreactions • Sometimescommonnamesareused,particularlyforthe digestionenzymessuchaspepsin andtrypsin • Somenamesdescribeboththesubstrateandthefunction • Forexample,alcoholdehydrogenase oxidesethanol ClassificationofEnzymes • Enzymesareclassifiedaccordingtothetypeofreactionthey catalyze: Class Reactionscatalyzed § Oxidoreductases Oxidation-reduction § Transferases Transfergroupsofatoms § Hydrolases Hydrolysis § Lyases § Isomerases § Ligases Addatoms/removeatomsto/froma doublebond Rearrangeatoms UseATPtocombinemolecules SystematicName • AccordingtotheInternationalunionof Biochemistryanenzymenamehastwoparts: - Firstpartisthenameofthesubstratesforthe enzyme. - Secondpartisthetypeofreactioncatalyzedby theenzyme.Thispartendswiththesuffix“ase”. Example:Lactatedehydrogenase 1 9/16/16 ECnumber ECnumbers Enzymesareclassifiedintosixdifferentgroups accordingtothereactionbeingcatalyzed.The nomenclaturewasdeterminedbytheEnzyme Commissionin1961(withthelatestupdate havingoccurredin1992),henceallenzymes areassignedan“EC” number.The classificationdoesnottakeintoaccount aminoacidsequence(ie,homology),protein structure,orchemicalmechanism. • ECnumbersarefourdigits,forexamplea.b.c.d, where“a” istheclass,“b” isthesubclass,“c” is thesub-subclass,and“d” isthesub-sub-subclass. The“b” and“c” digitsdescribethereaction, whilethe“d” digitisusedtodistinguishbetween differentenzymesofthesamefunctionbasedon theactualsubstrateinthereaction. EC1.Oxidoreductases • EC1.Oxidoreductases:catalyzethetransferof hydrogenoroxygenatomsorelectronsfrom onesubstratetoanother,alsocalledoxidases, dehydrogenases,orreductases.Notethat sincetheseare‘redox’ reactions,anelectron donor/acceptorisalsorequiredtocomplete thereaction. EC4.Lyases • EC4.Lyases – catalyzenon-hydrolytic(coveredin EC3)removaloffunctionalgroupsfrom substrates,oftencreatingadoublebondinthe product;orthereversereaction,i.e. additionof functiongroupsacrossadoublebond. • A-B→A=B+X-Y • Example:forAlcohol:NAD+oxidoreductase:EC numberis1.1.1.1 EC2.Transferases • EC2.Transferases – catalyzegrouptransfer reactions,excludingoxidoreductases (which transferhydrogenoroxygenandareEC1). Theseareofthegeneralform: • A-X+B↔BX+A EC5.Isomerases • EC5.Isomerases – catalyzesisomerization reactions,includingracemizations andcistransisomerizations. ClassificationofEnzymes TheSixClasses Enzymesareclassifiedaccordingtothetypeofreactiontheycatalyze: • • • • • • EC1.Oxidoreductases EC2.Transferases EC3.Hydrolases EC4.Lyases EC5.Isomerases EC6.Ligases Additionalinformationonthesubclasses,thesub-subclassesandsub-subsubclasses(ie,fullenzymeclassificationandnames)canbefoundatthe referencedweblink. • FromtheWebversion, http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html EC3.Hydrolases • EC3.Hydrolases– catalyzehydrolytic reactions.Includeslipases,esterases, nitrilases,peptidases/proteases.Theseareof thegeneralform: • A-X+H2 O↔X-OH+HA EC6.Ligases • EC6.Ligases-- catalyzesthesynthesisof various(mostlyC-X)bonds,coupledwiththe breakdownofenergy-containingsubstrates, usuallyATP XY • Includesdecarboxylasesandaldolases inthe removaldirection,andsynthasesintheaddition direction. 2 9/16/16 Oxidoreductases,Transferases andHydrolases Lyases,Isomerases andLigases Enzymeactionoverview • Enzymesarelargemoleculesthathaveasmallsection dedicatedtoaspecificreaction.Thissectioniscalled theactive site. • Theactivesitereactswiththedesiredsubstance,called thesubstrate. • Thesubstratemayneedanenvironmentdifferentfrom themostlyneutralenvironmentofthecellinorderto react.Thus,theactivesitecanbemoreacidicorbasic, orprovideopportunitiesfordifferenttypesofbonding tooccur,dependingonwhattypeofsidechainsare presentontheaminoacidsoftheactivesite. ActiveSiteofanEnzyme • Theactivesite isaregion withinanenzymethatfits theshapeofsubstrate molecules • Aminoacidside-chains aligntobindthesubstrate throughH-bonding,saltbridges,hydrophobic interactions,etc. • Productsarereleased whenthereactionis complete(theynolonger fitwellintheactivesite) ExampleofanEnzyme-CatalyzedReaction • Thereactionforthesucrase catalyzedhydrolysisofsucroseto glucoseandfructosecanbewrittenasfollows: E +S D ES ® E +P1 +P2 whereE =sucrase,S =sucrose,P1 =glucoseandP2 =fructose EnzymeSpecificity Enzyme-CatalyzedReactions • Enzymeshavevaryingdegreesofspecificity forsubstrates • Enzymesmayrecognizeandcatalyze: - asinglesubstrate - agroupofsimilarsubstrates - aparticulartypeofbond • Whenasubstrate(S)fitsproperlyinanactivesite,an enzyme-substrate(ES)complex isformed: E +S D ES • WithintheactivesiteoftheES complex,thereaction occurstoconvertsubstratetoproduct(P): ES ® E +P • Theproductsarethenreleased,allowinganother substratemoleculetobindtheenzyme - thiscyclecanberepeatedmillions(orevenmore)times perminute • Theoverallreactionfortheconversionofsubstrateto productcanbewrittenasfollows: E +S D ES ® E +P Isoenzymes • Isoenzymes aredifferentformsofanenzymethatcatalyze thesamereactionindifferenttissuesinthebody - theyhaveslightvariationsintheaminoacidsequences ofthesubunitsoftheirquaternarystructure • Forexample,lactatedehydrogenase(LDH),whichconverts lactatetopyruvate,consistsoffiveisoenzymes DiagnosticEnzymes • Thelevelsofdiagnosticenzymes inthebloodcanbeused todeterminetheamountofdamageinspecifictissues 3 9/16/16 Factorsthataffectenzymeaction DenaturingEnzymes (=conformationchange=changeofproteins’3Dstructure) • Whenanenzymeisdenatureditisdamaged. • Denaturingchangestheshape. • Withoutthecorrectshapeenzymeswon’t functionproperly. • HOWareenzymesdenatured? – Temperature – pH pHandEnzymeActivity • Enzymes aremostactiveatoptimumpH • Aminoacidswithacidicorbasicside-chainshavethe properchargeswhenthepHisoptimum • ActivityislostatloworhighpHastertiarystructureis disrupted EnzymeConcentrationandReactionRate • Therateofreactionincreasesasenzymeconcentration increases(atconstantsubstrateconcentration) • Athigherenzymeconcentrations,moreenzymesare availabletocatalyzethereaction(morereactionsatonce) • Thereisalinearrelationshipbetweenreactionrateand enzymeconcentration(atconstantsubstrateconcentration) Factorsthataffectenzymeaction Enzymesaremostlyaffectedbychangesintemperature andpH. TemperatureandEnzymeActivity • Enzymes aremostactiveatanoptimumtemperature(usually 37°Cinhumans) • Theyshowlittleactivityatlowtemperatures • Activityislostathightemperaturesasdenaturationoccurs • Toohighofatemperaturewilldenaturetheprotein components,renderingtheenzymeuseless. • pHrangesoutsideoftheoptimalrangewillprotonate ordeprotonate thesidechainsoftheaminoacids involvedintheenzyme’sfunctionwhichmaymake themincapableofcatalyzingareaction. OptimumpHforSelectedEnzymes • MostenzymesofthebodyhaveanoptimumpHofabout7.4 • However,incertainorgans,enzymesoperateatlowerand higheroptimumpHvalues SubstrateConcentrationandReactionRate • Therateofreactionincreasesassubstrateconcentration increases(atconstantenzymeconcentration) • Maximumactivity occurswhentheenzymeissaturated (whenallenzymesarebindingsubstrate) • Therelationshipbetweenreactionrateandsubstrate concentrationisexponential,andasymptotes(levelsoff) whentheenzymeissaturated Factorsthataffectenzymeaction Enzymesarealsoaffectedbytheconcentrationofsubstrate, cofactorsandinhibitors,aswellasallostericregulationand feedbackinhibition. • Theconcentrationof substratewilldictatehowmanyenzymes canreact.Toomuchsubstratewillslowtheprocessuntilmore enzymecanbemade. • Theavailabilityofcofactors alsodictateenzymeaction.Toolittle cofactorswillslowenzymeactionuntilmorecofactorsareadded. • Aninfluxofcompetitiveornon-competitiveinhibitorswillnot necessarilyslowtheenzymeprocess,butwillslowtheamountof desiredproduct. Enzymeactiontheories • LockandKey: Thistheory,postulatedby EmilFischerin1894,proposedthatan enzymeis“structurallycomplementary totheirsubstrates” andthusfittogether perfectlylikealockandkey.Thistheory formedthebasisofmostoftheideasof howenzymeswork,butisnotcompletely correct. 4 9/16/16 Enzymeactiontheories Lock-and-KeyModel • Inthelock-and-keymodel ofenzymeaction: - theactivesitehasarigidshape - onlysubstrateswiththematchingshapecanfit - thesubstrateisakeythatfitsthelockoftheactivesite • Thisisanoldermodel,however,anddoesnotworkforall enzymes InducedFitModel • Enzymescanformtotheshapeofits substrate. Enzymeactiontheories Enzymeactiontheories • InducedFit: Anenzymethatisperfectlycomplementarytoits substratewouldactuallynotmakeagoodenzymebecausethe reactionhasnoroomtoproceedtothetransitionstateofthe reaction. Togotocompletion,areactionmustgothroughthe transitionstate.Inthelockandkeytheory,thesubstrateorthe enzymecannotchangeconformationstothetransitionstate. Therefore,enzymesmustactuallybecomplementarytothe transitionstate sothereactionmayproceed.Thisideawas researchedbyHaldanein1930,andLinusPaulingin1946.This idealedtheInducedFittheory,postulatedbyDanielKoshland in1958,wheretheenzymeitself canchangeconformationsto facilitatethetransitionstateofthesubstrate.Thischangein conformationoftheenzymeallowsthenecessaryfunctional groupsattheactivesitetomoveclosertothesubstrate, enhancingtheefficiencyofthereaction. Enzymecofactors • Acofactor isasubstance,thatisnotaprotein,thatmust bindtotheenzymeinorderfortheenzymetowork. • metalionsascofactors-- Zn2+,Fe2+,Cu2+,others • Acofactorcanbeoforganicorigin.Anorganiccofactoris calledacoenzyme. InducedFitModel • Intheinduced-fitmodel ofenzymeaction: - theactivesiteisflexible,notrigid - theshapesoftheenzyme,activesite,andsubstrateadjust tomaximizethefit,whichimprovescatalysis - thereisagreaterrangeofsubstratespecificity • Thismodelismoreconsistentwithawiderrangeofenzymes Enzymecofactorscont. • Anenzymethatisbondedtoitscofactoriscalleda holoenzyme. • Anenzymethatrequiresacofactor,butisnotbondedto thecofactoriscalledanapoenzyme.Apoenzymes arenot activeuntiltheyarecomplexed withtheappropriate cofactor. • Cofactorsarenotpermanentlybonded.Permanently bondedcofactorsarecalledprostheticgroups. Coenzymes Coenzymes:smallermoleculesthataidinenzymechemistry. Enzymescan: a.Carryoutacid-basereactions b.Transientcovalentbonds c.Charge-chargeinteractions Enzymescannotdo: d.Oxidation- Reduction(=Redox)reactions e.Carbongrouptransfers Prostheticgroup:permanentlyassociatedwithanenzyme ortransientlyassociated. Holoenzyme:catalyticallyactiveenzymewithcofactor. Apoenzyme: Enzymewithoutitscofactor. Commoncoenzymes Manycoenzymesarederivedfromvitamins: • NAD + (nicotinamide adeninedinucleotide); derived fromniacin(B 3). • CoenzymeA(CoA);derivedfrompantothenicacid (B 5). • FAD(flavin adeninedinucleotide);derivedfrom riboflavin(B 2). Commom Coenzymes Coenzyme Reactionmediated Biotin Carboxylation Cobalamin (B12) Alkylationtransfers CoenzymeA Acyltransfers Flavin Oxidation-Reduction Lipoic acid Acyltransfers Nicotinamide Oxidation-Reduction Pyridoxal Phosphate Aminogrouptransfers Tetrahydrofolate One-carbongrouptransfers Thiaminepyrophosphate Aldehydetransfer 5 9/16/16 VitaminsareCoenzymeprecursors Thesearewatersolublevitamins.TheFatsolublevitaminsarevitaminsAand D. Commoncoenzymes Humanscannotsynthesizetheseandrelayontheirpresenceinourdiets. Thosewhohaveanunbalanceddietmaynotbereceivingasufficientsupply. Vitamin Coenzyme DeficiencyDisease Biotin Biocytin notobserved Cobalamin (B12) Cobalamin Perniciousanemia Folicacid tetrahydrofolate Neuraltubedefects Megaloblastic anemia Nicotinamide Nicotinamide Pantothenate CoenzymeA Pyridoxine(B6) Pyridoxal phosphate Riboflavin(B2) Thiamine(B1) Niacin(niacinamide)deficiencyleadstopellagracharacterizedbydiarrhea, dermatitisanddementia.PellagrawasendemicisSouthernUnitedStatesinthe early20thcentury.Niacincanbesynthesizedfromtheessentialaminoacid, tryptophan.Acorndietprevalentatthetimerestrictedtheabsorptionof tryptophancausingadeficiency.Treatmentofcornwithbasecouldreleasethe tryptophan(MexicanIndianstreatedcornwithCa(OH)2 beforemakingtortillas!) • ATP(adenosinetriphosphate);derivedfromNADHfrom carbohydratesconsumed.(ATPcanalsoactas neurotransmitter) • CTP(Cytidine triphosphate);derivedfromglutamateand carbamoylphosphate. • PAPS(3'-Phosphoadenosine-5'-phosphosulfate); derivedfrom adenosine5'-phosphosulfate(APS)andsulfateion. Pellagra Notobserved Notobserved Flavin Thiaminepyrophosphate Notobserved Beriberi Fattyacidsynthesis Coenzymereactions • Coenzymeshelptotransferafunctionalgrouptoamolecule. CoenzymeAisconvertedtoacetyl-CoenzymeA enzymeispyruvatedehydrogenase • Forexample,coenzymeA(CoA)isconvertedtoacetyl-CoAin themitochondriausingpyruvateandNAD+. • Acetyl-CoAcanthenbeusedtotransferanacetyl group (CH3CO)toaidinfattyacidsynthesis. • The“normal” wayanenzymefunctionsiswhenthespecific substratebindstotheactivesiteandcreatestheproducts. • Asimilarsubstratecanalsobondtotheactivesitecovalently andirreversibly.Thispreventstheenzymefromfunctioning. Irreversibleinhibition. • Asimilarsubstratecanbindtotheactivesite,notpermanently, andpreventsthedesiredsubstratefromenteringtheactivesite. Thischangestheproductsandfunctioningoftheenzyme.Thisis calledcompetitiveinhibition. Amoleculecanbondtoanotherpartoftheenzymeandcausea changeinconformation.Thischangecausestheactivesiteto changeshapeaswell.Thischangeinshapepreventsthedesired substratefromenteringtheactivesite.Thisiscallednoncompetitiveinhibition. Factorsthataffectenzymeaction • Enzymesthatcanbeactivatedwillbeaffectedbythe amountofactivatororinhibitorattachedtoits allostericsite.Anabundanceofanallostericactivator willconvertmoreenzymestotheactiveformcreating moreproduct. • Enzymesthatarepartofametabolicpathwaymaybe inhibitedbytheveryproducttheycreate.Thisis calledfeedbackinhibition.Theamountofproduct generatedwilldictatethenumberofenzymesusedor activatedinthatspecificprocess. EnzymeActivators Enzymeactivityandinhibition • Coenzymescanbederivedfromsourcesotherthanvitamins: • Chemicalsthathelptheenzymework. • Activatorsincreasetheenzymereactionrate. EnzymeInhibitors • Chemicalsthatpreventtheenzymefromworking. • Inhibitorsdecreasetheenzymereactionrate. Active Site Activator X Binding Site Substrate 6 9/16/16 EnzymeInhibitors • Inhibitors(I) aremoleculesthatcausealossof enzymeactivity • Theypreventsubstratesfromfittingintothe activesiteoftheenzyme: E+SD ES® E+P E+I D EI ® no Pformed ReversibleInhibitors(Non-competitiveInhibition) • Anon-competitiveinhibitor hasastructurethatis differentthanthatofthe substrate - itbindstoanallostericsite ratherthantotheactivesite - itdistortstheshapeofthe enzyme,whichaltersthe shapeoftheactivesiteand preventsthebindingofthe substrate • Theeffectcannotbereversed byaddingmoresubstrate, buteventuallyreversedby washingtheinhibitoraway. SummaryofEnzymes-I • • • • • • • • • Enzymesaremostlyproteins Theyarehighlyspecifictoareaction Theycatalyzemanyreactionsincludingbreakingdownnutrients,storingand releasingenergy,creatingnewmolecules,andcoordinatingbiological reactions. Enzymesuseanactivesite,butcanbeaffectedbybondingatotherareasof theenzyme. Someenzymesneedspecialmoleculescalledcofactorstocarryouttheir function. Cofactorsthatareorganicinnaturearecalledcoenzymes. Coenzymesareusuallyderivedfromvitamins. Coenzymestransferfunctionalgroupsfortheenzymetheyworkwith. EnzymesareaffectedbychangesinpH,temperature,theamountof substrate,cofactorsandinhibitors,aswellastheamountofallosteric inhibitorsandactivatorsandconcentrationofproductsthatcontrolfeedback inhibition. Reversible Inhibitors (CompetitiveInhibition) • Areversibleinhibitor goes onandoff,allowingthe enzymetoregainactivity whentheinhibitorleaves • Acompetitiveinhibitor is reversibleandhasa structurelikethesubstrate - itcompeteswiththe substratefortheactivesite - itseffectisreversedby increasingsubstrate concentration ExampleofaCompetitiveInhibitor • Malonate isacompetitiveinhibitorofsuccinatedehydrogenase - ithasastructurethatissimilartosuccinate - inhibitioncanbereversedbyaddingsuccinate IrreversibleInhibitors • Anirreversibleinhibitor destroysenzymeactivity,usuallyby bondingwithside-chaingroupsintheactivesite http://upload.wikimedia.org/wikipedia/commons/thumb/6/6f/Major_digestive_enzymes.png/750px-Major_digestive_enzymes.png Life Sciences-HHMI Outreach. Copyright 2009 President and Fellows of Harvard College TheSixClasses Biochemistry 3.2)Enzymes-II Introductiontoenzymestructureand function,andfactorsinvolvingtheir actionsandpathways Enzyme- Kinetics • • • • • • EC1.Oxidoreductases EC2.Transferases EC3.Hydrolases EC4.Lyases EC5.Isomerases EC6.Ligases Additionalinformationonthesubclasses,thesub-subclassesandsub-subsubclasses(ie,fullenzymeclassificationandnames)canbefoundatthe referencedweblink. • FromtheWebversion, http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html Prof. Dr. Klaus Heese 7 9/16/16 Some common types of enzymes Hydrolases Polymerases Nucleases Kinases Proteases Phosphatases Synthases ATP-ases Isomerases Oxidoreductases (dehydrogenases) Enzyme Active Sites Enzyme Active Sites Active sites: The region that binds substrate. Only a small fraction of the enzyme. Formed from AAs in different parts of the sequence. Active sites: Usually form a cleft or pocket. Substrates are bound by multiple weak interactions. carboxypeptidase pH,T Lock-Key-model Induced-Fit-Model Properties of Enzymes Reaction Thermodynamics • Enzymes are highly efficient and specific catalysts. • Enzymes stabilize transition states. How is Transition State Stabilization Achieved? • DGro is not changed ! Enzymes stabilize the transition state, lowering the activation barrier. How to Lower DG‡? Enzymes organizes reactive groups into proximity (---> induced-fit-model) – acid-base catalysis: give and take protons – covalent catalysis: change reaction paths – metal ion catalysis: use redox cofactors, pKa shifters • Uncatalyzed bimolecular reactions: – electrostatic catalysis: preferential interactions with • Uncatalyzed unimolecular reactions: transition state EA • The enzyme lowers the activation barrier (EA or DG‡ ) compared to the uncatalyzed reaction. • Enzymes alter rates, not equilibria. • Reaction rates depend on concentrations of enzymes, substrates, and on the efficiency of the enzyme. Rate Acceleration two free reactants ® single restricted transition state conversion is entropically unfavorable flexible reactant ® rigid transition state conversion is entropically unfavorable for flexible reactants • Catalyzed reactions: Enzyme uses the binding energy of substrates to organize the reactants to a fairly rigid ES complex Entropy cost is paid during binding Rigid reactant complex ® transition state conversion is entropically OK EA Enzymes stabilize the transition state, lowering the activation barrier. Enzyme Kinetics Kinetics is the study of the rates of reactions • Enzymes endow cells with the remarkable capacity to exert kinetic control over thermodynamic potentiality • Enzymes are the agents of metabolic function • What we want to be able to determine: – Maximum velocity – Substrate affinity – Inhibitor affinity • What it can tell us: – Flow through metabolic pathways – Utilization of substrates • What can we do with the information: – Control and manipulate metabolic events 8 9/16/16 Enzyme Kinetics Keq = Enzymatic Equations II Enzymatic Equations reminder: Consider: [A] + [B] Enzyme Kinetics Enzyme Kinetics Reaction Thermodynamics [C] + [D] E+S ES EP E+S E+S E+P There are at least three steps……. Keq depends only on the nature of the products and the reactants. E+P E+P Km = (k-1+ kcat )/k1 usually kcat <<< k-1 , so Km = k-1 /k1 = Kd The Michaelis-Menten Equation Kinetics: Vmax and Km Important Conclusions of Michaels Menten Kinetics kcat Vmax = the maximum reaction rate. Km = substrate concentration where V = Vmax /2 Measures affinity of enzyme for substrate. • when [S] >> KM, the equation reduces to • when [S] << KM, the equation reduces to The Michaelis-Menten equation • Ideal Rate: Enzyme Kinetics Reaction rate (V) varies with substrate concentration. Effect of Substrate Concentration • when [S]= KM, the equation reduces to Enzyme Kinetics Enzyme Kinetics ES kcat =[E][S]=(k-1+kcat )/k1 [ES]--->with[E]total=[E]t and[S]0 =[S]attimepoint0: [E]=[E]t –[ES]--->([E]t –[ES])[S]=Km [ES]--->[ES]=[S][E]/Km +[S]---> istheproductformationcontrollingthevelocityofthereaction:---> V0 =kcat [ES]=kcat {[S][E]/Km +[S]};if[S]highsothatenzymefullysatisfied:Vmax =kcat [E]t --->V0 =Vmax [S]/Km +[S] Reaction will proceed spontaneously only when the change in free energy (DG) is negative. V = Vmax k-1 EP atsteadystate:forward=reversereaction:d[ES]/dt =-d[ES]/dt <--->k1 [E][S]=k-1+kcat [ES] [C][D] [A][B] [S] Km + [S] ES k1 v= Vmax [ S ] Km + S • Deviations due to: – Limitation of measurements – Substrate inhibition – Substrate prep contains inhibitors – Enzyme prep contains inhibitors Effect of Substrate Concentration Vmax and Km Different substrates, Vmax and Km Enzyme Kinetics Vmax [S] V= K--------------M +[S] Enzyme Kinetics Enzyme Kinetics Vmax Vmax dependsontheamountofenzyme. Km is a property of both enzyme and substrate. 9 9/16/16 • A plot of 1/V versus 1/[S] will give a straight line with slope of KM/Vmax and y intercept of 1/Vmax • Such a plot is known as a Lineweaver-Burk double reciprocal plot v0 1 v0 1 v0 = = ([S]+Km ) Vmax [S] = Enzyme inhibitors are important for a variety of reasons 3) they can be used to gain information about the regulation or control of a metabolic pathway. 4) they can be very important in drug design. The“normal” wayanenzymefunctionsiswhenthespecific substratebindstotheactivesiteandcreatestheproducts. • Asimilarsubstratecanalsobondtotheactivesitecovalently andirreversibly.Thispreventstheenzymefromfunctioning. Irreversibleinhibition. • Asimilarsubstratecanbindtotheactivesite,notpermanently, andpreventsthedesiredsubstratefromenteringtheactivesite. Thischangestheproductsandfunctioningoftheenzyme.Thisis calledcompetitiveinhibition. • Amoleculecanbondtoanotherpartoftheenzymeandcausea changeinconformation.Thischangecausestheactivesiteto changeshapeaswell.Thischangeinshapepreventsthedesired substratefromenteringtheactivesite.Thisiscallednoncompetitiveinhibition. = [S] Vmax [S] Km 1 Vmax [S] + + Km Vmax [S] 1 Vmax V and [S] can be determined experimentally Enzyme inhibition Tool to study enzymatic reactions. Important in host/pathogen interactions. Important in drug design. Irreversible (suicide) inhibition (eg - nerve gas). Reversible inhibition: competitive (eg. - transition state analogues). non-competitive. uncompetitive. Type of inhibition can be determined experimentally. EnzymeActivators Enzymeactivityandinhibition • Vmax [S] ([S]+Km ) Linearized double-reciprocal plot is good for analysis of two-substrate data or inhibition. Lineweaver – Burk Double Reciprocal Plots Enzyme Inhibition 1) they can be used to gain information about the shape on the enzyme active site and the amino acid residues in the active site. 2) they can be used to gain information about the chemical mechanism. Significance of Km Determination of Kinetic Parameters • Chemicalsthathelptheenzymework. • Activatorsincreasetheenzymereactionrate. Enzyme Kinetics Enzyme Kinetics Lineweaver – Burk Double Reciprocal Plots • It is difficult to determine Vmax experimentally • The equation for a hyperbola can be transformed into the equation for a straight line by taking the reciprocal of each side • The formula for a straight line is y = mx + b • Km is a constant • Small Km means tight binding; high Km means weak binding • Useful to compare Km for different substrates for one enzyme Hexokinase : D-fructose – 1.5 mM D-glucose – 0.15 mM • Useful to compare Km for a common substrate used by several enzymes Hexokinase: D-glucose – 0.15 mM Glucokinase: D-glucose – 20 mM Inhibition Patterns Inhibitors act in a variety of mechanisms • An inhibitor may bind at the same site as one of the substrates – these inhibitors structurally resemble the substrate • An inhibitor may bind at an alternate site affecting catalytic activity without affecting substrate binding • Many inhibitors do both • Most common types – Competitive – Mixed or Non-competitive – Uncompetitive EnzymeInhibitors • Chemicalsthatpreventtheenzymefromworking. • Inhibitorsdecreasetheenzymereactionrate. Active Site Activator X Binding Site Substrate 10 9/16/16 EnzymeInhibitors • Inhibitors(I) aremoleculesthatcausealossof enzymeactivity • Theypreventsubstratesfromfittingintothe activesiteoftheenzyme: E+SD ES® E+P E+I D EI ® no Pformed Competitive Inhibition ReversibleInhibitors(CompetitiveInhibition) • Areversibleinhibitor goes onandoff,allowingthe enzymetoregainactivity whentheinhibitorleaves • Acompetitiveinhibitor is reversibleandhasa structurelikethesubstrate - itcompeteswiththe substratefortheactivesite - itseffectisreversedby increasingsubstrate concentration Competitive Inhibition ExampleofaCompetitiveInhibitor • Malonate isacompetitiveinhibitorofsuccinatedehydrogenase - ithasastructurethatissimilartosuccinate - inhibitioncanbereversedbyaddingsuccinate Competitive Inhibition • Unimolecular Reaction • A competitive inhibitor reduces the amount of free enzyme available for substrate binding thus increasing the Km for the substrate • Bimolecular • The effect of a competitive inhibitor can be overcome with high concentrations of the substrate ReversibleInhibitors(Non-competitiveInhibition) • Anon-competitiveinhibitor hasastructurethatisdifferent thanthatofthesubstrate - itbindstoanallostericsite ratherthantotheactivesite - itdistortstheshapeofthe enzyme,whichaltersthe shapeoftheactivesiteand preventsthebindingofthe substrate • Theeffectcannotbereversed byaddingmoresubstrate,but eventuallyreversedbywashing theinhibitoraway. Reaction Change of Lineweaver – Burk graph by enzyme inhibition Mixed or Non-Competitive Inhibition Non-Competitive • The inhibitor can bind to both free enzyme and the ES complex • The affinity of the inhibitor to the two complexes might be different – If binding of inhibitor changes the affinity for the substrate, Km will be changed and called mixed inhibition – If only Vmax affected called Non-competitive inhibitor Change of Lineweaver – Burk graph by enzyme inhibition 11 9/16/16 Mixed Inhibition Mixed Inhibition • • The result will be decrease in Vmax and either an increase or decrease in Km The effect of an non-competitive inhibitor can only be partially overcome by high concentrations of the substrate Uncompetitive Inhibition Uncompetitive Reactionoftheirreversibleinhibitor diisopropylfluorophosphate (DFP)witha serineprotease; DFPisairreversiblecholinesteraseinhibitor: Diisopropyl fluorophosphate isaverypotent neurotoxin.ItsLD50inratsis1.3mg/kg.It combineswiththeaminoacidserineatthe activesiteoftheenzyme acetylcholinesterase, anenzymethat deactivatestheneurotransmitter acetylcholine.Neurotransmittersareneeded tocontinuethepassageofnerveimpulses fromoneneurontoanother(ortothe (striated)muscle)acrossthesynapse.Once theimpulsehasbeentransmitted, acetylcholinesterase functionstodeactivate theacetylcholinealmostimmediatelyby breakingitdown.Iftheenzymeisinhibited, acetylcholineaccumulatesandnerve impulsescannotbestopped,causing prolongedmusclecontraction.Paralysis occursanddeathmayresultsincethe respiratorymusclesareaffected. Uncompetitive Inhibition • An uncompetitive inhibitor binds to the enzyme substrate complex but not to free enzyme • The result is a decrease in Vmax and Km • The effect of an uncompetitive inhibitor can not be overcome by high concentrations of the substrate IrreversibleInhibitors • Anirreversibleinhibitor destroysenzymeactivity,usuallyby bondingwithside-chaingroupsintheactivesite SummaryofEnzymes-II • • • • • • • • • Enzymesaremostlyproteins Theyarehighlyspecifictoareaction Theycatalyzemanyreactionsincludingbreakingdownnutrients,storingand releasingenergy,creatingnewmolecules,andcoordinatingbiological reactions. Enzymesuseanactivesite,butcanbeaffectedbybondingatotherareasof theenzyme. Someenzymesneedspecialmoleculescalledcofactorstocarryouttheir function. Cofactorsthatareorganicinnaturearecalledcoenzymes. Coenzymesareusuallyderivedfromvitamins. Coenzymestransferfunctionalgroupsfortheenzymetheyworkwith. EnzymesareaffectedbychangesinpH,temperature,theamountof substrate,cofactorsandvarioustypesofinhibitors. 12